Clinical Chemistry Exam 1 Lecture 5

Kjeldahl
Classic method for quantitating total protein
Kjeldahl =
Total Nitrogen Content x 16%
Biuret
Routine method for quantitating total protein
Biuret process
Cupric ions + peptide bonds = Violet
Required 2 or more peptide bonds and an alkaline medium
Color is proportional to protein concentration
For Biuret you should read color on a spec at ____ nm
540
Should you avoid hemolysis with Biuret
Yes
Dye-Binding is often associated with which type of substance?
Albumin
Albumin + dye absorbs light at about _____ nm
600
True or False:
Absorbance is directly proportional to albumin concentration with Dye-Binding testing method
True
Dye Binding Principle
Positive charged Albumin + Negative charged dye
What types of dyes are associated with Dye-Binding?
Bromcresol green
Bromcresol purple
Methyl Orange
Nephelometry
Serum proteins added to specific antibody
Detectors measure light scatter caused by ag-ab complexes
Amount of light is proportional to the protein’s concentration
What substances are often associated with Nephelometry
Tf
Hapt
Immunoglobulins
C’
Is Serum Protein Eelectrophoresis quantitative or semi-quantitative?
Semi-quantitative
With the SPE in what direction will negatively charged proteins travel?
Toward the anode (+)
What does the rate of migration with the SPE depend on?
Size
Charge
Shape
Time
Support Media
Endosmosis
What are the support media associated with SPE?
Cellulose acetate
Agarose gel
Polyacrylamide gel
Stains used with SPE?
Ponceau S
Amido Black
Coomassie Blue
A _____ converts visible protein bands into peaks. The area under each peak can be semi-quantitated.
Densitometer
Monoclonal gammopathy
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Polyclonal gammopathy
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Hypogammaglobulinemia
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Alpha 1-Antitrypsin deficiency
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Beta-Gamma bridging
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Nephrotic Syndrome
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Normal SPE
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Proliferation of one clone of immunoglobulin
Monoclonal gammopathy
What diseases are associated with Monoclonal Gammopathy
Multiple Myeloma
Waldenstrom’s Macroglobulinemia
True or False:
SPE does not identify the specific protein
True
What test would you run after SPE?
IFE
True or False:
M spike may appear in alpha 2, beta, or gamma regions
True
What type of SPE are Osteolytic lesions and elevated calcium often associated with?
Monoclonal Gammopathy
Increase in all gamma proteins
Polyclonal gammopathy
What illnesses are often associated with Polyclonal Gammopathy
Chronic inflammation
Infections
Hypogammaglobulinemia is associated with what type of illness?
Immunodeficiency
Is Hypogammaglobulinemia inherited or acquired?
Both
How can Hypogammaglobulinemia be acquired?
Cancer, chemo, HIV, etc.
What illness is associated with Alpha 1-Antitrypsin deficiency?
Emphysema
Beta-gamma bridging is associated with what type of gamma proteins?
Fast-moving
What illness is Beta-gamma bridging associated with?
Liver cirrhosis
In Nephrotic syndrome what substance is associated with increased protein loss through the kidneys?
Acute phase reactants
In Nephrotic syndrome what substance is associated with decreased protein loss through the kidneys?
Albumin
Globulin =
Total protein – Albumin
Normal Ratio of A/G
1.5 to 1
Low A/G ratio
Too many globulins or Too little albumin
High A/G ratio
Not enough globulins
What illnesses are associated with: low A/G ratio due to too many globulins
MM
Autoimmune diseases
What illnesses are associated with: low A/G ratio due to too little albumin
Renal disease
Liver disease
What illnesses are associated with: not enough globulins
Genetic
Leukemia
Immunosuppressed
Immunofixation Electrophoresis
Identifies the specific protein causing the M spike on SPE or UPE
Normal Urine protein electrophoresis
Urine concentrated x 100
Normal Urine protein electrophoresis shows only a _____ amount of albumin
small
Urine with Bence Jones Protein
Obvious M Spike
IFE needed to identify type of light chain present
What type of band is more obvious with a Normal CSF Electrophoresis?
prealbumin
CSF with Oligoclonal Bands
Abnormal banding in the gamma region
IgG Index >0.8
What disease is CSF with Oligoclonal Bands consistent with?
Multiple Sclerosis
Which type of Electrophoresis is fast and requires little sample?
Capillary (Zone) Electrophoresis
With Capillary Electrophoresis, when the capillary is connected to a power supply, the charged molecules migrate at different rates depending on what?
net charge
Capillary Electrophoresis: ____ are measured by absorbance as they pass through a detector
Proteins
Plasma Viscosity
Non-specific measure of protein changes due to inflammation/tissue damage
Does Plasma Viscosity Increase or Decrease in parallel to the ESR
Increase
Is Plasma Viscosity affected by hematocrit or testing delays?
No
What diseases is an increased plasma viscosity associated with?
Myeloma
Hyperfibrinogenemia
How is Plasma Viscosity measured with?
Viscometer
What are the reporting units of Plasma Viscosity?
Centipoises (cP or cps)
What is the Normal Rate of Plasma Viscosity?
1.1-1.5 cP
Water at Room Temperature is about ___ cP
1
What is the Critical Value for Plasma Viscosity?
>3.0 cP
If G is increased or A is decreased the ratio will be low. What 3 diseases are associated with this?
MM
Chronic Disease
Nephrotic Syndrome
If G is decreased the ratio will be high. What disease is associated with this?
Immune Suppression
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